Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
  • G01N33/56988—HIV or HTLV
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/974—Aids related test

Definitions

• the invention relates to a method for the determination of antibodies against HIV and a reagent suitable therefor.
• HIV antibodies The determination of HIV antibodies is an important diagnostic task.
• Cyclic HIV-2 and HIV-1 peptides are also known from EP-A 0 326 490. These peptides must also be used in large quantities for an immunological test and result in very high blank values.
• the object of the present invention is therefore to provide a method for the determination of HIV antibodies with which these disadvantages can be eliminated.
• a biotinylated polypeptide which contains the amino acid sequence CAFRQVC is preferably used for the determination of HIV 2 antibodies.
• streptavidin solid phase is described in EP-A 0 344 578, the content of which is the subject of the present application.
• Suitable polypeptide sequences are described in WO 88/08005, EP-A 0 292 454, EP-A 0 283 327, EP-A 0 326 490.
• the sequences listed there can be used according to the invention after biotinylation, cyclization and isolation of the biotinylated monomeric cyclic peptides.
• biotinylated peptides are compounds of the formulas: XA-NSWGCAFRQVCHTT or NSWGCAFRQVCH-BY, where X is a biotinylaminocaproic acid, biotinylamniobutyric acid, biotinyl, N-epsilon-biotinyllysine, N-epsilon (biotinylaminocaproyl) lysine, N-delta-biotinylornithine or N-delta (biotinylaminocaproyl) ornithine residue; Y is an N-epsilon-biotinyllysine, N-epsilon (biotinylaminocaproyl) lysine, N-delta-biotinylornithine or N-delta (biotinylaminocaproyl) ornithine residue; A is a bond or peptide sequence such as
• biotinylated peptides are compounds of the formulas: XC-GCSGKLICTT-D or C-GCSGKLICTT-DY where C is a bond, KDQQLLGIW, DQQLLGIW, QQLLGIW, QLLGIW, LLGIW, LGIW, GIW, IW, or W; D a bond, AVPWNAS, AVPWNA, AVPWN, AVPW, AVP, AV or A; can be and X and Y have the meaning given above.
• biotinylated polypeptides of which the first peptide has the amino acid sequence CAFRQVC and the second peptide the amino acid sequence CSGKLIC. It is also preferred to use a peptide which contains both amino acid sequences.
• biotinylated peptides can be prepared, for example, by synthesis of the peptides according to Merrifield JACS 85 (1964), 2146. Biotinylation can be carried out, for example, according to PNAS 80 (1983), 4045.
• the peptides are cyclized by oxidation before or after biotinylation and the monomer is isolated by chromatography, preferably HPLC chromatography.
• the peptides can be prepared by first synthesizing the corresponding peptide sequences, then cyclizing the peptides to cystine peptides by oxidation of the cysteine SH groups and then using a common biotinylating agent, e.g. Biotinylaminocaproonic acid N-hydroxysuccinimdester biotinylated, or by first biotinylating and then cyclizing the peptides.
• a common biotinylating agent e.g. Biotinylaminocaproonic acid N-hydroxysuccinimdester biotinylated
• the peptides can be produced, for example, by solid phase synthesis; eg with temporary tert. Butyloxycarbonyl protection of the alpha amino acids (Merrifield, JACS 85 (1964), 2146) or with temporary fluorenylmethoxycarbonyl protection ("Fmoc”) (Meienhofer, Int.J. Pept.Prot.Res 11 (1978), 246).
• solid phase synthesis eg with temporary tert. Butyloxycarbonyl protection of the alpha amino acids (Merrifield, JACS 85 (1964), 2146) or with temporary fluorenylmethoxycarbonyl protection (“Fmoc”) (Meienhofer, Int.J. Pept.Prot.Res 11 (1978), 246).
• Another possibility for the production of the peptides according to the invention is the introduction of the biotin residue during solid phase synthesis, e.g. by using N-alpha-Fmoc-N-epsilon (biotinylaminocaproyl) lysine, N-alpha-Fmoc-N-epsilon-biotinyllysine or, in the case of biotinylation of the N-terminal amino acid, biotin-N-hydroxysuccinimester or biotinylaminocaproic acid N-hydroxysuccinimester.
• Another particularly preferred production method is the cyclization of the peptides after the solid phase synthesis on the polymeric support.
• cysteine protected by the trityl residue is used on the SH group and the solid-phase-bound peptides are treated with iodine in a solvent mixture which contains a perfluorinated compound, preferably dichloromethane / hexafluoroisopropanol.
• a perfluorinated compound preferably dichloromethane / hexafluoroisopropanol.
• the biotinylated monomeric peptides are preferably used in a concentration of 0.005-0.4 ⁇ g / ml, particularly preferably in a concentration of 0.02-0.3 ⁇ g / ml.
• the length of the peptides is a maximum of 50 amino acids, preferably a maximum of 35 and particularly preferably a maximum of 20 amino acids.
• An antibody which is directed against the Fc ⁇ part of human immunoglobulin is preferably used as the labeled receptor.
• This antibody can be both monoclonal and polyclonal.
• An antibody is preferably used which is directed against the Fc ⁇ part of human IgG.
• markings familiar to the person skilled in the art, such as, for example, enzymes (for example peroxidase, alkaline phosphatase), radionucleotides, fluorogenic or chromogenic substrates, cofactors, coloidal gold or magnetic particles can be used as markings.
• enzymes for example peroxidase, alkaline phosphatase
• radionucleotides for example fluorogenic or chromogenic substrates, cofactors, coloidal gold or magnetic particles
• cofactors for example, coloidal gold or magnetic particles
• the method according to the invention is carried out by adding the sample and biotinylated antigen to a streptavidin solid phase, incubating and washing. Labeled antibody against HIV 1 and / or HIV 2 antibody is then added, incubated, washed and the label in the solid phase or the separated liquid phase is determined.
• a variant of the determination method consists in that polypeptides labeled as labeled receptors, which consist of a maximum of 50 amino acid residues and contain the amino acid sequence CAFRQVC and / or CSGKLIC, preferably CAFRQSV, the two cysteines (C) of the amino acid sequences being cyclized to form cystine bridges and the polypeptide is monomeric can be used.
• Peptides of the formulas are preferably used: XA-NSWGCAFRQVCHTT or NSWGCAFRQVCH-BY, where X is a biotinylaminocaproic acid, biotinylamniobutyric acid, biotinyl, N-epsilon-biotinyllysine, N-epsilon (biotinylaminocaproyl) lysine, N-delta-biotinylornithine or N-delta (biotinylaminocaproyl) ornithine residue; Y is an N-epsilon-biotinyllysine, N-epsilon (biotinylaminocaproyl) lysine, N-delta-biotinylornithine or N-delta (biotinylaminocaproyl) ornithine residue; A is a bond or peptide sequence such as QARL, ARL,
• biotinylated peptides are compounds of the formulas: XC-GCSGKLICTT-D or C-GCSGKLICTT-DY where C is a bond, KDQQLLGIW, DQQLLGIW, QQLLGIW, QLLGIW, LLGIW, LGIW, GIW, IW, or W; D a bond, AVPWNAS, AVPWNA, AVPWN, AVPW, AVP, AV or A; can be and X and Y have the meaning given above.
• the peptide AsnSer (tBu) TrpGlyCys (Trt) AlaPheArg (Mtr) GlnValCys (Trt) His (Trt) Thr (TBu) Thr (tBu) is synthesized on the solid phase.
• the peptide-resin is then shaken in a 100-fold volume with iodine-saturated dichloromethane / hexafluoroisopropanol (3: 1) for 2 hours and washed three times with dichloromethane, dimethylformamide and isopropanol.
• HIV-2 antibodies are determined in a 2-step sandwich immunoassay.
• the reagents with the following composition are used for detection:
• biotinylaminocaproyl-NSWGCAFRQVCHTT (peptide 1, preparation according to Example 1) 40 mmol / l phosphate buffer pH 7.0 0.9% by weight of table salt 10 vol.% Bovine serum albumin

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Virology (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Organic Chemistry (AREA)
• Urology & Nephrology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• AIDS & HIV (AREA)
• Gastroenterology & Hepatology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)

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• 1989
  • 1989-01-23 DE DE3901857A patent/DE3901857A1/de not_active Withdrawn
• 1990
  • 1990-01-17 DE DE59008779T patent/DE59008779D1/de not_active Expired - Fee Related
  • 1990-01-17 AT AT90100871T patent/ATE120549T1/de not_active IP Right Cessation
  • 1990-01-17 EP EP90100871A patent/EP0379949B1/fr not_active Expired - Lifetime
  • 1990-01-19 US US07/467,602 patent/US5001049A/en not_active Expired - Lifetime
  • 1990-01-23 JP JP2011992A patent/JP2559870B2/ja not_active Expired - Fee Related

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